Stable transgenesis and targeted gene insertion have many potential applications in both gene therapy and cell engineering. However, current strategies are often inefficient and non-specifically insert the transgene into prokaryotic or eukaryotic genomic DNA. The inability to control the location of genome insertion can lead to highly variable levels of transgene expression throughout the population due to position effects within the genome. Additionally, current methods of stable transgenesis and amplification of transgenes often result in physical loss of the transgene, transgene silencing overtime, insertional mutagenesis by the integration of a gene and autonomous promoter inside or adjacent to an endogenous gene, the creation of chromosomal abnormalities and expression of rearranged gene products (comprised of endogenous genes, the inserted transgene, or both), and/or the creation of vector-related toxicities or immunogenicity in vivo from vector-derived genes that are expressed permanently due to the need for long-term persistence of the vector to provide stable transgene expression.
Lentiviral and retroviral vectors have been used for gene integration and can stably integrate their viral genomes along with an encoded transgene into the host genome of transduced cells via integrase. See, e.g., U.S. Pat. Nos. 5,994,136; 6,165,782; and 6,428,953. However, transgene expression can be highly variegated depending on where the virus integrates in the host genome, transgene silencing can occur over time, insertional mutagenesis can occur, and the site of integration can not be controlled. Furthermore, the number of transgene copies inserted into the genome can only be somewhat controlled by the dose of the vector added to the cells. Also, if the viral vector expresses additional, non-native genes, these can be toxic to the cells or can cause an immune response that leads to the destruction of the transduced cell in vivo.
In eukaryotic cells, stable transgenesis can also be achieved using a recombinant DNA sequence encoding a selection marker along with the transgene of interest. Cells that stably express the transgene of interest can be isolated by selecting for stable expression of the selection marker. The number of transgene copies and the level of expression can be amplified by prolonged selection using drugs such as methotrexate (DHFR gene) or PALA (CAD gene). However, this approach still suffers from position effects and can not allow for the targeting of where genes can be inserted into the genome. (Coquelle et al. (1997) Cell 89:215-225). In addition, the amplification frequency is low (typically <10E-4) and often requires selection to occur for 20 cell generations with gradually increasing drug concentration and amplification using a single selection step is extremely inefficient (Tlisty et al. (1989) Proc. Nat'l. Acad. Sci. USA 86: 9441-9445; Kempe et al. (1976) Cell 9:541-550; Singer et al. (2000) Proc. Nat'l. Acad. Sci. USA 97:7921-7926). Furthermore, amplification can only be carried out in tumor cells and does not work in primary cells (Tlisty (1990) Proc. Nat'l. Acad. Sci. USA 87:3132-3139; Wright et al. (1990) Proc. Nat'l. Acad. Sci. USA 87:1791-1795) and, in many human and rat cell lines, the amplification protocol can lead to the formation of unstable extrachromosomal double minutes instead of homogenous repeats of the chromosomal region encoding the selection marker and transgene of interest and/or to a greater occurrence of chromosomal instability and rearrangements in tumor cells (Pauletti et al. (1990) Proc. Nat'l. Acad. Sci. USA 87:2955-2959; Smith et al. (1997) Proc. Nat'l. Acad. Sci. USA 94:1816-1821; Fougere-Deschatrette et al. (1982) in Gene Amplification, ed. Schimke, R. T. (Cold Spring Harbor Lab. Press, Plainview, N.Y., pp. 29-32; Singer et al. (2000) Proc. Nat'l. Acad. Sci. USA 97:7921-7926). Stable, high transgene expression also often requires continued exposure to the selection drug (Schimke, R. T. (1984) Cell 37:705-713; Stark et al (1984), Annual Rev. Biochem., 53:447-503; Tlisty et al. (1989) Proc. Nat'l. Acad. Sci. USA 86:9441-9445). Thus, given the requirement for prolonged exposure to drugs, increase in chromosomal instability, and inability to use this method for primary cells, marker selection is not applicable in the engineering of cells for cellular therapies.
Single or multiple copies of a transgene can also be stably integrated into cells via artificial chromosomes or stable episomes. These systems can replicate and remain stable in mammalian cells, contain large gene payloads that can include genomic regulatory elements and generally do not integrate to cause insertional mutagenesis (Conese et al (2004) vol. 11, pp. 1735-1741). However, there have been issues with their overall stability over time including, for example, loss and/or rearrangement of these artificial chromosomes as well as an increase in the integration of the artificial chromosomes or episomes into the native chromosomes over time, which can cause de-stabilization of the native chromosome(s) (e.g. creation of a dicentric chromosome (Suzuki N. et al (2006), JBC, vol. 281, pp. 26615-26623; Shinohara T. et al (2000) Chromosome Res., vol. 8, pp. 713-725; Ohzeki J., et al (2002) J. Cell Biol, vol. 159, pp. 765-775; Grimes B R, et al. (2002) Mol Ther, Vol. 5, pp. 798-805; Nakano M, (2003) J Cell Sci, vol. 116, pp. 4021-4034). Furthermore, artificial chromosomes and stable episomes function only in dividing cell lines or proliferating primary cells where DNA-based delivery (e.g. electroporation or cationic lipids) works efficiently (Suzuki N. et al (2006), JBC, vol. 281, pp. 26615-26623) and segregation errors occur that cause instability and loss over time (Rudd M K, et al (2003) Mol Cell Bio, Vol. 23, pp. 7689-7697). In addition, the isolation and maintenance of cell clones containing these stable episomes or artificial chromosomes require selection-based methods and, accordingly, have all the problems detailed above. Finally, safety concerns are raised by the fact that these stable episomes are often derived from self-replicating viral-based vectors (e.g. EBV or bovine papapilloma virus) that have been shown to persist extrachromosomally in mammalian cells, but require the expression of an oncogenic viral-derived transgene such as EBNA1.
Zinc finger nucleases can be used to efficiently drive targeted gene insertion at extremely high efficiencies using a homologous donor template to insert novel gene sequences into the break site via homology-driven repair (HDR). See, for example, United States Patent Publications 20030232410; 20050208489; 20050026157; 20050064474; and 20060188987, and International Publication WO 2007/014275, the disclosures of which are incorporated by reference in their entireties for all purposes. This does not require long-term persistence of the delivery vector, avoiding issues of insertional mutagenesis and toxicities or immunogenicity from vector-derived genes.
However, there remains a need for controlled, site-specific integration of a single or multiple copies of a transgene to allow for higher or lower, but stable and uniform, transgene expression within a cell population. There also remains a need for targeted gene integration that does not result in variegated transgene expression, insertional mutagenesis caused by position effects or chromosomal instability related to transgene amplification.